Antimicrobial susceptibility profiling and genomic diversity of Acinetobacter baumannii isolates: A study in western Iran.

Background and Objective Acinetobacter baumannii is an aerobic non-motile Gram-negative bacterial pathogen that is resistant to most antibiotics. Carbapenems are the most common antibiotics for the treatment of infections caused by this pathogen. Mechanisms of antibiotic-resistance in A. baumannii are mainly mediated by efflux pumps-lactamases. The aim of this study was to determine antibiotic susceptibility, the possibility of existence of OXAs genes and fingerprinting by Pulsed-Field Gel Electrophoresis (PFGE) among clinical isolates of Acinetobacter collected from Kermanshah hospitals. Materials and Methods One hundred and four isolates were collected from patients attending Imam Reza, Taleghani and Imam Khomeini hospitals of Kermanshah (Iran). Isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics was assessed with Kirby-Bauer disk diffusion method. PCR was performed for detection of bla OXA-23, bla OXA-24, bla OXA-51 and bla OXA-58 beta-lactamase genes. Clonal relatedness was estimated by PFGE (with the restriction enzyme Apa I) and DNA patterns were analyzed by Gel compare II 6.5 software. Results All isolates showed high-level of resistance to imipenem, meropenem as well as to other antimicrobial agents, while no resistance to polymyxin B, colistin, tigecylcine and minocycline was observed. The bla OXA-23like and bla OXA-24 like were found among 77.9% and 19.2% of the isolates, respectively. All isolates were positive for bla OXA-51, but none produced any amplicon for bla OXA-58. PFGE genotype analysis suggested the existence of eight clones among the 104 strains [A (n = 35), B (n = 29), C (n = 19), D (n = 10), E (n = 4), F (n = 3), G (n = 3), H (n = 1)]. Clone A was the dominant clone in hospital settings particularly infection wards so that the isolates in this group, compared to the other clones, showed higher levels of resistance to antibiotics. Conclusion The bla OXA-51-like and bla OXA-23like were the predominant mechanisms of resistance to imipenem in A. baumannii. A high prevalence of clone A, B and C in different parts of the healthcare system showed that hospitalized patients should be safeguarded to prevent the spread of these clones. Early recognition of the presence of carbapenem-resistant A. baumannii clones is useful for preventing their spread within the hospital environment.


INTRODUCTION
Acinetobacter baumannii is an aerobic non-motile Gram-negative coccobacillus and polymorphic bacterial pathogen that is easily spread from one patient to others, persisting in the environment for many days (1). It is the third most common pathogenic bacteria isolated from hospitalized patients with pneumonia like with PCR. Furthermore, in this study, we report an evaluation of CHDL-producing A. baumannii isolates collected from various Hospitals of Kermanshah and the association between their susceptibility and genetic profile as well as PFGE typing for total isolates. Pulsed-Field Gel Electrophoresis (PFGE) was performed to investigate the genetic relation among the isolates and determined wide spread clone (11).
MBL and eSBL screening. The isolates were identified for a phenotypic MBL screening with the E-test MBL (bio-Mérieux, Marcy L'Etoile, France) as per the manufacturer's instructions (14). Indistinguishable MBL producing isolates were detected and confirmed by Cica-Beta-Test Strip (Kanto Chemical, Tokyo) as per the manufacturer's instructions.
Screening for ESBL-producing organisms was carried out using Double Disk Synergy Test (DDST). Those strains that showed resistance to imipenem by disk diffusion test were re-checked in T-test (15). PCR amplification of OXA genes. PCR screening was performed for the carbapenemase-encoding genes (bla OXA-23-like , bla OXA-24-like , bla OXA-51-like and bla  ). PCR analysis was performed using the primers described by Kuo et al. (16).

Pulsed-field gel electrophoresis (PFGe). All
A. baumannii isolates were analyzed by CHEF Mapper PFGE according to the protocols previously described by Durmaz et al. with little modifications (17). We used A. baumannii ATCC 19606 for normalization of the gel (17, 18). The solution absorption coefficient was set at 600 nm. The cell lysis solution I consisted of Tris-HCl 50 mM (pH 8), EDTA 50 mM (pH 8), 1 mg/ml proteinase K and 1.5 mg/ml lysozyme (Roche Applied Sciences) that was kept shaking in water bath at 37°C for 45 min. Lysis solution II consisted of EDTA 0.5 M (pH 8), 0.4 mg/ml proteinase K and 1% sarcosyl was transferred to tubes containing plugs while kept shaking in water bath at 50°C for 2 h. Digestion of all organisms were performed with ApaI (New England Biolabs) restriction enzyme. One half of each plug was transferred to 2 ml tubes that contains 100 μl buffer 1X consisting of ApaI enzyme, and then was incubated at 25°C for 2 h. electrophoresis. 1% (w/v) of pulsed-field Megabase Agaros (Bio-Rad laboratories) added into 100 ml of TBE 0.5X (1 liter of 5X stock solution with pH 8). DNA separation was performed in TBE 0.5X (pH 8) buffer in a pulsed-field electrophoresis system (CHEF Mapper; Bio-Rad Laboratories, Hercules, CA, USA) by program two state with the following conditions: temperature 14°C; voltage 6 V/cm; switch angle, 120°; switch ramp 2.2-35 s for 19 h. We used Lambda Ladder PFGE Marker (NEB: N0340) as molecular size marker. The Dice coefficient was used to calculate similarities, and the unweighted paired group method based on average linkages (UPGMA) was used for cluster analysis with Gel compare II version 6.5 (Applied Maths, St Martens-Latem, Belgium) and with a high similar pattern (similarity > 87%) were considered to be derived from a cluster (closely related strains) (17-19).
Statistical analysis. Data were recorded and entered into a database. Statistical analyses were performed using Stata (Version 11.0). Continuous variables were compared using one-way analysis of variance. Variables were analyzed by chi-square test. A P-value of < 0.05 was set as the level of statistical significance for all analyses in the study.

ReSULTS
A total of 104 clinical isolates of A. baumannii were collected from 3 hospitals in Kermanshah (Iran). The   results showed high-level of resistance to imipenem (79.8%) and meropenem (75%) as well as resistance to other antibiotics (Table 1). Resistance was observed against polymyxin B (13.5%), minocycline (16.3%), colistin (10.6%). Resistance rates for tigecylcine was low (2.9%). 84 isolates (80.8%) produced MBLs. In our study, 85.6% (n = 89) of isolates were able to produce carbapenemase that was significantly associated with imipenem and meropenem resistance (p value < 0.001). Furthermore, 32.7% (n = 34) of isolates show-ed multidrug resistance phenotype, (MDR, resistant to third generation cephalosporins, aminoglycosides and fluoroquinolone) and 8.7% (n = 9) pandrug resistant (PDR, resistant to all available antibacterial agents except polymyxin B and colistin) phenotype, whereas none of the isolates was extensively drug resistant phenotype (XDR). XDR has been defined for the strains showing resistance to some of the most effective anti-bacterial drugs (20). Using PCR assay 77.9% and 19.2% of the isolates were positive for bla OXA-23like and bla OXA-24 like genes, respectively. While all isolates were positive for bla OXA-51like , none gave any amplicon for the bla OXA-58 like . Co-existence of bla OXA-23 and bla OXA-24 like was observed among 16.4% of the isolates. The corelations between carbapenemase and bla OXA-23 like was statistically significant (P value 0.002).
According to Tenover's criteria(18), a major PFGE type (clone A) containing 35 isolates was identified in 3 studied hospitals in this study. A subtyped designated as A1 was found for type A that differed in migration of one to three bands (Fig. 1). Other PFGE patterns include types B (n = 29), C (n = 19), D (n = 10), E (n = 4), F (n = 3), G (n = 3) and H (n = 1)]. The types A, B, C and D were the dominant types found in the hospitals. Type H consisted of single isolate. The PFGE analysis revealed that 18 isolates were closely related. All iso-lates from infection wards belong to clone A ( Table 2). All isolates within type F (100%) showed high-level of resistance to imipenem followed by type A (91.4%) (P value 0.03). In addition, 97.1% of the isolates with type A (n = 89) were able to produce carbapenemase (P value 0.023) ( Table 2)

DISCUSSION
Carbapenem resistance has been increasingly common issue among A. baumannii isolates in Iranian hospitals in recent years. This study was aimed to evaluate the molecular epidemiology of carbapenemresistant A. baumannii in Kermanshah hospitals, with the aim of identifying predominant clonal circulating. Our study showed low susceptibility rates to the most clinically available antimicrobial agents for treatment of A. baumannii-induced infections. There was a resistance to polymyxin B, colistin, minocycline, whereas low resistance to tigecylcine (Table 1). This antibiotic can be helpful in treating A. baumannii-related infections in hospital settings. This study reports one of the first large outbreaks of MDR 32.7% A. baumannii in west of Iran. The global incidence of meropenem resistance in A. baumannii was approximately 6% in 1998 but it has dramatically increased to approximately 29% in 2005 (10). This rapid increase has also been happened in the Kermanshah hospitals, where resistance rates to imipenem and meropenem were 79.8% and 75%, respectively. Findings of the present study showed that susceptibility to tigecylcine in 82 out of 83 (98.7%) isolates was significant. A similar increase in the resistance of A. baumannii isolates to imipenem and meropenem was found in the previous studies conducted in Iran during 2006Iran during -2010. In addition, A. baumannii isolates (obtained in 2006-2007) from Singaporean hospitals were also highly resistant to carbapenems (22). All the clinical A. baumannii isolates obtained in 2006-2007 from Malaysia, exhibited high resistance to all the examined antimicrobial agents except for polymyxin B (Different mechanisms are involved in the A. baumannii resistance to imipenem. β-Lactamase is important factor to carbapenem-resistance (23). The acquisition of carbapenem resistance in A. baumannii is mainly because of the production of two types of β-lactamases: Metallo-β-lactamases (MBLs) and carbapenem-hydrolysing class D β-lactamases (CHDLs). The CHDLs such as bla OXA-23 , bla OXA-24 , bla OXA-51 appear to be more prevalent and important to carbapenem resistance in this bacterium in some countries including Bahrain, UAE and Kuwait (10). However, the bla OXA-23-like and bla OXA-24-like genes were responsible for the majority of carbapenem resistance in the isolates studied in this research.
Carbapenemase were found in 89 strains (85.6%), including 74 (83.2%) bla OXA-23like that the bla OXA-23like was associated with carbapenemase (P = 0.002) because the bla OXA-23like gene is associated with Tn2006 that is capable of transposing from bacteria to bacteria could be located on the plasmid or a chromosome (24). bla OXA-23like was associated with imipenem (P 0.002) and also with meropenem (P http://ijm.tums.ac.ir < 0.001). In conclusion, bla OXA-51-like and bla OXA-23like were the predominant mechanisms of resistance to imipenem in A. baumannii. However, for a global epidemiologic analysis, further studies in large scale and different places should be conducted.
Various molecular typing systems have been developed so far to facilitate better understanding of epidemiology of infection with A. baumannii (11). This is the first published study of PFGE typing among the clinical strains of A. baumannii conducted in west of Iran. We obtained 8 clones and 20 sub clone. Clone A was involved in the majority of outbreaks in Kermanshah. It occurred at different hospitals wards and was the predominant pattern 100% cultured from Infectious Disease wards. Clone B was the second most common pattern involved in outbreaks. Isolates within this clone were mainly positive for bla OXA-23like where clone A was dominant for the presence of this gene. The clone (A, A1) were associated with cefotaxime (P value 0.004) indicating a close relationship with spreading dominant clone. Most of MDR and PDR phenotypes were presented in the clones A, B and C. Clonal outbreaks of A. baumannii-induced infections containing different OXA-type carbapenemases were reported in Brazil, Taiwan, Iran, Spain, Malaysia, Italy, Turkey, Korea and Argentina (5,16,[20][21][25][26]. It is conjectured that these genes are closely associated with outbreaks in some countries. Similarly, the results of this study support the viewpoint indicating clonal spread as the main reason for the increasing trend of imipenem, meropenem resistance as well as other antibiotics studied in the hospitals of this study. It is possible that the transfer of these clones to other wards by staff or hospital equipments. A high prevalence of the clone A, B and C in different parts of the healthcare system showed that hospitalized patients should be highly careful to prevent the spread of these clones. Finally, all isolates collected from the hospital 3 in Kermanshah contained bla OXA-23like , and all isolates belonged to the clone A; therefore, this clone was responsible for the outbreak in this hospital. Early recognition of the presence of carbapenem-resistant A. baumannii clones is useful for preventing their spread within the hospital environment.